Automated multi-attribute method sample preparation using high-throughput buffer exchange tips.

RATIONALE: The multi-attribute method (MAM) has become a valuable mass spectrometry (MS)-based tool that can identify and quantify the site-specific product attributes and purity information for biotherapeutics such as monoclonal antibodies (mAbs) and fusion molecules in recent years. As we expand the use of the MAM at various stages of drug development, it is critical to enhance the sample preparation throughput without additional chemical modifications and variability. METHODS: In this study, a fully automated MAM sample preparation protocol is presented, where rapid desalting in less than 15 minutes is achieved using miniaturized size-exclusion chromatography columns in pipette tips on an automated liquid handler. The peptide samples were analyzed using an electrospray ionization (ESI) orbitrap mass spectrometer coupled to an ultra-high-performance liquid chromatography (UHPLC) system with a dual column switching system. RESULTS: No significant change was observed in product attributes and their quantities compared with manual, low-artifact sample preparation. The sample recovery using the buffer exchange tips was greatly enhanced over the manual spin cartridges while still demonstrating excellent reproducibility for a wide variety of starting sample concentrations. Unlike a plate desalting system, the individual columns provide flexibility in the number of samples prepared at a time and sample locations within plates. CONCLUSIONS: This automated protocol enables the preparation of up to 96 samples with less "at-bench" time than the manual preparation of a smaller batch of samples, thereby greatly facilitating support of process development and the use of the MAM in quality control.

Nanotopographical Cues Mediate Osteogenesis of Stem Cells on Virus Substrates through BMP-2 Intermediate.

Recent studies have demonstrated rapid osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) on substrates with plant virus modified nanotopographical cues as a promising strategy for bone repair; however, the mechanisms remain unclear. We hypothesized that the highly structurally ordered virus coat proteins, responsible for targeting specific cellular components, are critical for the osteogenesis promotion. In this study, hybrid viral gold nanorods were prepared to explore the effects of highly ordered arranged virus coat proteins on osteogenic differentiation of BMSCs. The results herein indicate that it is the nanotopographical cues modified by structurally ordered virus nanoparticles, not the chemical properties of virus surface, that mediate osteogenesis. Bone morphogenetic protein 2 (BMP-2) expression is significantly increased and serves as a modulator that mediates the osteogenic differentiation in response to the viral particle coatings. After BMP-2 is inhibited by Noggin, the osteogenesis promoting effects are significantly compromised, demonstrated by lower alkaline phosphatase activity and calcium sequestration. This study reveals that plant virus modified nanotopographical substrates promote osteogenic differentiation of BMSCs through increasing BMP-2 autocrine. It provides key insights to engineering functional materials for rapid bone repair.

Effect of Roughness on in Situ Biomineralized CaP-Collagen Coating on the Osteogenesis of Mesenchymal Stem Cells.

Because of its outstanding osteo-conductive property, a calcium phosphate (CaP) coating has been used as an implant coating for bone tissue engineering. Nevertheless, the issues, such as harsh fabrication conditions, long-term stability and biocompatibility, and the requirement for expensive instruments, still exist in current coating techniques. To address these issues, the CaP coatings doped with collagen (CaP-Col) were in situ generated on polyelectrolyte multilayers (PEMs) by incubating PEMs in a mixture of the collagen, phosphate, and calcium ions. The resulting coatings have controllable physical properties (chemical composition, crystallinity, and roughness) and good stability before and after incubation with cell culture medium. We also found that both the cellular viability and osteogenesis of mesenchymal stem cells (MSCs) were closely related to the roughness of PEMs/CaP-Col, one of the easily ignored physical factors in current coating designs but very critical. The existed roughness window (between 18 ± 1.2 and 187 ± 7.3 nm) suitable for MSC proliferation on PEMs/CaP-Col coating and the optimal roughness (∼98 ± 3.5 nm) for MSC osteogenesis further demonstrated that the roughness was a critical factor for bone formation. Therefore, we envision that our exploration of the effects of surface roughness on MSC behaviors would provide better guidance for the future design of material coating and eventual medical success.

Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream uses.

The continued acceleration of time-to-market product development and rising demand for biotherapeutics have hastened the need for higher throughput within the biopharmaceutical industry. Automated liquid handlers (ALH) are increasingly popular due to flexible programming that enables processing of multiple samples with an array of functions. This flexibility is useful in streamlining research that requires chromatographic procedures to achieve product purity for downstream analysis. However, purification of biologics often requires additional off-deck buffer exchange steps due to undesirable elution conditions such as high acid or high salt content. Expanding the capability of ALHs to perform purification in sequence with buffer exchange would, therefore, increase workflow efficiency by eliminating the need for manual intervention, thus expediting sample preparation. Here we demonstrate two different automated purifications using pipet-based dispersive solid-phase extraction (dSPE). The first is an affinity purification of His-tagged proteins from bacterial lysate. The second is an anion-exchange purification of plasmid DNA. Both methods are followed by buffer exchange performed by an ALH. Percent recoveries for the three purified recombinant proteins ranged from 51 ± 1.2 to 86 ± 10%. The yields were inversely correlated to starting sample load and protein molecular weight. Yields for plasmid purification ranged between 11.4 ± 0.8 and 13.7 ± 0.9 µg, with the largest plasmid providing the highest yield. Both programs were rapid, with protein purification taking <80 min and plasmid purification <60 min. Our results demonstrate that high-quality, ready-to-use biologics can be obtained rapidly from a crude sample after two separate chromatographic processes without manual intervention.

Multivalent ligand displayed on plant virus induces rapid onset of bone differentiation.

Viruses are monodispersed biomacromolecules with well-defined 3-D structures at the nanometer level. The relative ease to manipulate viral coat protein gene to display numerous functional groups affords an attractive feature for these nanomaterials, and the inability of plant viruses to infect mammalian hosts poses little or no cytotoxic concerns. As such, these nanosized molecular tools serve as powerful templates for many pharmacological applications ranging as multifunctional theranostic agents with tissue targeting motifs and imaging agents, potent vaccine scaffolds to induce cellular immunity and for probing cellular functions as synthetic biomaterials. The results herein show that combination of serum-free, chemically defined media with genetically modified plant virus induces rapid onset of key bone differentiation markers for bone marrow derived mesenchymal stem cells within two days. The xeno-free culture is often a key step toward development of ex vivo implants, and the early onset of osteocalcin, BMP-2 and calcium sequestration are some of the key molecular markers in the progression toward bone formation. The results herein will provide some key insights to engineering functional materials for rapid bone repair.

Porous alginate hydrogel functionalized with virus as three-dimensional scaffolds for bone differentiation.

In regenerative medicine, a synthetic extracellular matrix is crucial for supporting stem cells during its differentiation process to integrate into surrounding tissues. Hydrogels are used extensively in biomaterials as synthetic matrices to support the cells. However, to mimic the biological niche of a functional tissue, various chemical functionalities are necessary. We present here, a method of functionalizing a highly porous hydrogel with functional groups by mixing the hydrogel with a plant virus, tobacco mosaic virus (TMV), and its mutant. The implication of this process resides with the three important features of TMV: its well-defined genetic/chemical modularity, its multivalency (TMV capsid is composed of 2130 copies of identical subunits), and its well-defined structural features. Previous studies utilizing the native TMV on two-dimensional supports accelerated mesenchymal stem cell differentiation, and surfaces modified with genetically modified viral particles further enhanced cell attachment and differentiation. Herein we demonstrate that functionalization of a porous alginate scaffold can be achieved by the addition of viral particles with minimal processing and downstream purifications, and the cell attachment and differentiation within the macroporous scaffold can be effectively manipulated by altering the peptide or small molecule displayed on the viral particles.

Factors Compromising Glucuronidase Performance in Urine Drug Testing Potentially Resulting in False Negatives.

Next generation ß-glucuronidases can effectively cleave glucuronides in urine at room temperature. However, during the discovery studies, additional challenges were identified for urine drug testing across biologically relevant pH extremes and patient urine specimens. Different enzymes were evaluated across clinical urine specimens and commercially available urine control matrices. Each enzyme shows distinct substrate preferences, pH optima, and variability across clinical specimens. These results demonstrate how reliance on a single glucuronidated substrate as the internal hydrolysis control cannot ensure performance across a broader panel of analytes. Moreover, sample specific urine properties compromise ß-glucuronidases to varying levels, more pronounced for some enzymes, and thereby lower the recovery of some drug analytes in an enzyme-specific manner. A minimum of 3-fold dilution of urine with buffer yields measurable improvements in achieving target pH and reducing the impact of endogenous compounds on enzyme performance. After subjecting the enzymes to pH extremes and compromising chemicals, one particular ß-glucuronidase was identified that addressed many of these challenges and greatly lower the risk of failed hydrolyses. In summary, we present strategies to evaluate glucuronidases that aid in higher accuracy urine drug tests with lower potential for false negatives.

Enhancing the multi-attribute method through an automated and high-throughput sample preparation.

The multi-attribute method (MAM), a recent advance in the application of liquid chromatography-mass spectrometry within the pharmaceutical industry, enables the simultaneous monitoring of multiple product quality attributes in a single analytical method. While MAM is coupled with automated data processing and reporting, the sample preparation, based on proteolytic peptide mapping, remains cumbersome and low throughput. The standard sample preparation for MAM relies on protein denaturation, reduction, and alkylation prior to proteolytic digestion, but often a desalting step is required to maintain enzymatic activity. While most of the sample preparation can be automated on a standard robotic liquid handling system, a streamlined approach for protein desalting and temperature modulation is required for a viable, fully automated digestion. In this work, for the first time, a complete tip-based MAM sample preparation is automated on a single robotic liquid handling system, leveraging a deck layout that integrates both heating and cooling functionalities. The fully automated method documented herein achieves a high-throughput sample preparation for MAM, while maintaining superior method performance.Abbreviations: MAM: multi-attribute method; PQAs: product quality attributes; CE: capillary electrophoresis; IEX: ion-exchange chromatography; HILIC-FLR: hydrophilic interaction liquid chromatography coupled to a fluorescence detector; RP-LC/UV: reversed-phase liquid chromatography coupled to a UV detector; MS: mass spectrometry; NPD: new peak detection; GdnHCl: guanidine hydrochloride; TIC: total ion current; pAb: polyclonal antibody; IgG: immunoglobulin G; DTT: dithiothreitol; IAA: iodoacetic acid; TFA: trifluoroacetic acid; A280: absorbance at 280 nm wavelength; 96MPH: 96-channel multi-probe head; CPAC: Cold Plate Air Cooled; HHS: Hamilton Heater Shaker; DWP: Deep-Well Plate; PCR: Polymerase Chain Reaction; NTR: Nested Tip Rack; Met: methionine; Trp: tryptophan; N-term pQ: N-terminal glutamine cyclization; Lys: lysine; PAM: peptidylglycine α-amidating monooxygenase; G0F: asialo-, agalacto-, bi-antennary, core substituted with fucose; G1F: asialo-, mono-galactosylated bi-antennary, core substituted with fucose; G2F: asialo-, bi-galactosylated bi-antennary, core substituted with fucose; G0: asialo-, agalacto-, bi-antennary; Man5: oligomannose 5; Man8: oligomannose 8; TriF: asialo-, tri-galactosylated tri-antennary, core substituted with fucose.

Variants of glycosyl hydrolase family 2 ß-glucuronidases have increased activity on recalcitrant substrates.

Glucuronidated drug metabolites can be quantified from urine samples by first hydrolyzing conjugates with ß-glucuronidase (ß-GUS) and then separating free drug molecules by liquid chromatography and mass spectrometry detection (LC-MS). To improve the activity and specificity of various ß-GUS, we designed enzyme chimeras and generated site-saturation variants based on structural analyses, then screened them for improved activity on drug metabolites important to clinical and forensic drug-testing laboratories. Often, an increase of activity on one substrate of interest was countered by loss of activity against another, and there was no strong correlation of activity on standard ß-glucuronidase substrates to activity on recalcitrant drug glucuronides. However, we discovered a chimera of two enzymes from different species of Aspergillus that displays a 27 % increase in activity on morphine-3-glucuronide than the parent proteins. Furthermore, mutations in the M-loop, which is a loop near the active site, resulted in numerous variants with dramatically increased rates of hydrolysis on drug glucuronides. Specifically, the M-loop variant Q451D/A452E of a ß-GUS from Brachyspira pilosicoli has a 50-fold and 25-fold increase in activity on the recalcitrant substrates codeine-6-glucuronide and dihydrocodeine-6-glucuronide, respectively, compared to the parent enzyme.

Comparison of Purified ß-glucuronidases in Patient Urine Samples Indicates a Lack of Correlation Between Enzyme Activity and Drugs of Abuse Metabolite Hydrolysis Efficiencies Leading to Potential False Negatives.

Pain management laboratories analyze biological fluids (urine, saliva or blood) from patients treated for chronic pain to ensure compliance and to detect undisclosed drug use. The quantitation of multi-panel drugs in urine and tissues utilizes ß-glucuronidase to cleave the glucuronic acid and liberate the parent drug for mass spectrometry analysis. This work focuses on the comparison of three different, purified and commercially available ß-glucuronidases across 83 patient urine samples. One enzyme is genetically modified, expressed in bacteria and the other two enzymes are purified from abalone. The results indicate that the source of ß-glucuronidase plays an important role in substrate specificity which in turn dictates hydrolysis efficiency. Contaminants in the enzyme solutions also interfere with analyte detection. Altogether, these factors impact precision and accuracy of data interpretation, leading up to 13% positive/negative disagreement.

Increased activity of ß-glucuronidase variants produced by site-directed mutagenesis.

ß-glucuronidase (BGus) is an essential glycosyl hydrolase which has been widely used in biological and biomedical applications. In this paper, we report the construction and screening of nineteen Escherichia coli BGus (EBGus) mutants using site-directed mutagenesis. The mutants G559N, G559S and G559T showed a 3-5 fold increase in enzyme activity in comparison to wild type EBGus. In particular, G559S, with the highest activity, showed 2-6 fold enhanced activity compared to abalone and snail BGus extracts. Moreover, the glycine to serine mutagenesis for the same site in Staphylococcus sp. RLH1 BGus (StBGus) exhibited significantly enhanced activity, which indicated the importance of the G559→S mutation on BGus function. Based on this structural analysis, we postulate that the mutation at G559 plays an important role in the stabilization of the enzyme conformation, and thereby facilitates the effective binding of substrate.

Influence of Surface Topographical Cues on the Differentiation of Mesenchymal Stem Cells in Vitro.

Adult stem cell research has been advanced in recent years because of the cells' attractive abilities of self-renewal and differentiation. Topography of materials is one of the key features that can be harnessed to regulate stem cell behaviors. Stem cells can interact with underlying material through nanosized integrin receptors. Therefore, the manipulation of topographical cues at a nanoscale level can be employed to modulate the cell fate. In this review, we focus our discussion on the different surface topographical cues, especially, with an emphasis on the viral nanoparticle-coated materials, and their effects on stem cell differentiation.

Degradation of Opioids and Opiates During Acid Hydrolysis Leads to Reduced Recovery Compared to Enzymatic Hydrolysis.

Drug monitoring laboratories utilize a hydrolysis process to liberate the opiates from their glucuronide conjugates to facilitate their detection by tandem mass spectrometry (MS). Both acid and enzyme hydrolysis have been reported as viable methods, with the former as a more effective process for recovering codeine-6-glucuronide and morphine-6-glucuronide. Here, we report concerns with acid-catalyzed hydrolysis of opioids, including a significant loss of analytes and conversions of oxycodone to oxymorphone, hydrocodone to hydromorphone and codeine to morphine. The acid-catalyzed reaction was monitored in neat water and patient urine samples by liquid chromatography-time-of-flight and tandem MS. These side reactions with acid hydrolysis may limit accurate quantitation due to loss of analytes, possibly lead to false positives, and poorly correlate with pharmacogenetic profiles, as cytochrome P450 enzyme (CYP2D6) is often involved with oxycodone to oxymorphone, hydrocodone to hydromorphone and codeine to morphine conversions. Enzymatic hydrolysis process using the purified, genetically engineered ß-glucuronidase (IMCSzyme®) addresses many of these concerns and demonstrates accurate quantitation and high recoveries for oxycodone, hydrocodone, oxymorphone and hydromorphone.

Virus Nanoparticles Mediated Osteogenic Differentiation of Bone Derived Mesenchymal Stem Cells.

There are few methodologies that allow manipulating a biomaterial surface at nanometer scale, which controllably influence different cellular functions. In this study, virus nanoparticles with different structural features are selected to prepare 2D substrates with defined nanoscale topographies and the cellular responses are investigated. It is demonstrated that the viral nanoparticle based substrates could accelerate and enhance osteogenesis of bone derived mesenchymal stem cells as indicated by the upregulation of osteogenic markers, including bone morphogenetic protein-2, osteocalcin, and osteopontin, at both gene and protein expression levels. Moreover, alkaline phosphatase activity and calcium mineralization, both indicators for a -successful bone formation, are also increased in cells grown on these nanoscale possessed substrates. These discoveries and developments present a new paradigm for nanoscale engineering of a biomaterial surface.

RGD-conjugated rod-like viral nanoparticles on 2D scaffold improve bone differentiation of mesenchymal stem cells.

Viral nanoparticles have uniform and well-defined nano-structures and can be produced in large quantities. Several plant viral nanoparticles have been tested in biomedical applications due to the lack of mammalian cell infectivity. We are particularly interested in using Tobacco mosaic virus (TMV), which has been demonstrated to enhance bone tissue regeneration, as a tunable nanoscale building block for biomaterials development. Unmodified TMV particles have been shown to accelerate osteogenic differentiation of adult stem cells by synergistically upregulating bone morphogenetic protein 2 (BMP2) and integrin-binding bone sialoprotein (IBSP) expression with dexamethasone. However, their lack of affinity to mammalian cell surface resulted in low initial cell adhesion. In this study, to increase cell binding capacity of TMV based material the chemical functionalization of TMV with arginine-glycine-aspartic acid (RGD) peptide was explored. An azide-derivatized RGD peptide was "clicked" to tyrosine residues on TMV outer surface via an efficient copper(I) catalyzed azide-alkyne cycloaddition (CuAAC) reaction. The ligand spacing is calculated to be 2-4 nm, which could offer a polyvalent ligand clustering effect for enhanced cell receptor signaling, further promoting the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs).

Polyvalent display of RGD motifs on turnip yellow mosaic virus for enhanced stem cell adhesion and spreading.

Turnip yellow mosaic virus (TYMV) is a stable 28 nm icosahedral plant virus that can be isolated in gram quantities. In order to study the polyvalent effect of Arg-Gly-Asp (RGD) clustering on the response of bone marrow stem cells (BMSCs), an RGD motif was genetically displayed on the coat protein of the TYMV capsid. Composite films composed of either wild-type TYMV or TYMV-RGD44, in combination with poly(allylamine hydrochloride) (PAH), were fabricated by a layer-by-layer adsorption of virus and PAH. The deposition process was studied by quartz crystal microbalance, UV-visible spectroscopy and atomic force microscopy. BMSC adhesion assays showed enhanced cell adhesion and spreading on TYMV-RGD44 coated substrates compared to native TYMV. These results demonstrate the potential of TYMV as a viable scaffold for bioactive peptide display and cell culturing studies.

A plant virus substrate induces early upregulation of BMP2 for rapid bone formation.

Many nanoscale materials have been developed to investigate the effects on stem cell differentiations via topographical and chemical cues for applications in tissue engineering and regenerative medicine. The use of plant viruses as cell supporting substrates has been of particular interest due to the rapid induction of bone marrow derived mesenchymal stem cells (BMSCs) towards osteogenic cells. In this study, the role of Tobacco mosaic virus (TMV) and its early effects on osteoinduction with particular emphasis on the regulation of bone morphogenetic protein-2 (BMP2) was examined. We observed that the cells on the virus substrate immediately aggregated and formed bone-like nodules within 24 hours. An immediate increase in BMP2 gene and protein expression for cells on the TMV substrate was observed within 8 hours of osteoinduction. Moreover, BMP2 expression was highly localized to cells within the cell aggregates. This enhanced differentiation only occurred when TMV was coated on a solid support but not upon adding the virus to the media solution. Taken together, the results from this study highlight the potential of virus-based nanomaterials to promote endogenous BMP2 production which may prove to be a unique approach to studying the regulatory mechanisms involved in early osteoblastic differentiation.